Bluetongue (BT) is an arthropod-borne infectious viral disease of ruminants. Cattle and goats may be readily infected with the causative BTV but without extensive vascular injury and therefore these species generally fail to show pronounced clinical signs. In contrast, the disease in sheep is characterized by catarrhal inflammation of the mucous membranes of the mouth, nose and forestomachs, and by inflammation of the coronary bands and laminae of the hoofs. There is an excoriation of the epithelium, and ultimately necrosis of the buccal mucosa; the swollen and inflamed tongue and mouth can take on a blue color from which the disease is named (Spreull 1905). The mortality rate in sheep is estimated at 1-30%.
BTV is the prototype virus of the Orbivirus genus (Reoviridae family) and is made up of at least 24 different serotypes(Wilson and Mecham 2000). Different strains of BTV have been identified world-wide throughout tropical and temperate zones. BTV infection has occurred as far as 45° N in Europe, as far as 50° N in Asia and North America, and as far South as 35°. BTV is not contagious between ruminants thus the distribution of BTV is dependent on the presence of arthropod vector species of coides sp. (biting midges), with different vector species occurring in different regions of the world. Recent data suggests that genetic drift and founder effect contribute to diversification of individual gene segments of field strains of BTV (Bonneau, Mullens et al. 2001). It has been shown that BTV seropositive animals are resistant to reinfection with the homologous BTV serotype.
BTV infection of ruminants is transient, while infection of the Culicoides insect vector is persistent. The duration of viremia depends on the animal species and the strain of BTV. It has been reported that viremia can be very transient in sheep and may last for up to 41 days in BTV-infected individuals, up to 42 days in goats, and up to 100 days in cattle. Since BTV infection of cattle often results in prolonged but not persistent viremia, cattle serve as a reservoir from which virus may be ingested by the Culicoides vector and then transmitted to other ruminants (Anderson, Stott et al. 1985; MacLachlan 1994; MacLachlan and Pearson 2004). The ecology of many species of Culicoides vectors is poorly understood and their breeding sites are largely uncharacterized, and their rates of dispersal unknown. Culicoides sonorensis is the principal vector of BTV in North America. Female Culicoides insects become persistently infected with BTV and can transmit the virus after an extrinsic incubation period of up to 14 days (Mullens, Tabachnick et al. 1995). BTV overwintering in temperate zones may occur through vertically infected insect vectors, although recent data indicates that there is reduced expression of the outer capsid genes during persistent BTV infection in larval stages of the insect vectors (White, Wilson et al. 2005).
The virions of BTV have a diameter of ˜69 nm with a double-shelled coat (capsid) that sometimes is surrounded by a lipoprotein “pseudo-envelope” derived from the cell membranes of infected cells. The BTV genome includes 10 distinct segments of double-stranded RNA that collectively encode seven structural (VP1 through VP7) and four non-structural (NS1, NS2, NS3 and NS3a) proteins (Roy 1996); 9 of the genome segments are monocistronic whereas segment 10 encodes both NS3 and NS3A using a second, inframe initiation codon. Genomic RNA is encapsidated in the icosahedral virion particle by a double layered protein capsid (Verwoerd, Els et al. 1972). The icosahedral core consists of two major (VP3 and VP7) and three minor proteins (VP1, VP4, VP6) and is surrounded by the outer capsid which consists of VP2 and VP5 that respectively are encoded by genomic segments 2 and 5 (Roy 1996). VP2 is responsible for binding and entry of BTV into cells, neutralization, serotype-specificity and hemagglutination. Multimeric forms of VP2 (dimers and trimers) decorate much of the surface of a VP5 scaffold on the outer surface of viral particles (Hassan and Roy 1999). VP2 varies most amongst the 24 BTV serotypes, and levels of anti-VP2 antibody correlate with virus neutralization in vitro and in vivo (Huismans and Erasmus 1981). VP5 also varies markedly between different serotypes and strains of BTV (de Mattos, de Mattos et al. 1994; DeMaula, Bonneau et al. 2000) and although no VP5-specific neutralizing MAb's have been identified to date, data suggests that this protein has a role in neutralization and serotype determination through its conformational influence on VP2 (Huismans and Erasmus 1981; Roy, Urakawa et al. 1990; DeMaula et al., 2000). Purified VP2, immunoadsorbed with BTV anti-core serum to remove trace amounts of VP7, was injected into sheep. An initial dose of 50 micrograms of VP2 was sufficient to induce VP2-precipitating antibodies as well as neutralizing and hemagglutination-inhibiting antibodies. These sheep were fully protected against challenge with a virulent strain of the same BTV serotype. Lower doses of VP2 still provided a significant level of protection even though no neutralizing antibodies were not detected prior to challenge (Huismans, van der Walt et al. 1987). Recent results show that VP2 and NS1 express epitopes recognized by cytotoxic T-lymphocytes (CTL) (Andrew, Whiteley et al. 1995) while it is unlikely that VP7 and VP5 have CTL epitopes. So far, VP3, VP4, VP6, NS2 and NS3 have not stimulated a CTL response in sheep (Lobato, Coupar et al. 1997) Table 1 (modified from, (Wilson and Mecham 2000)), below summarizes the genes of BTV and their protein function:
TABLE 1Bluetongue virus genes and encoded proteins with location, properties, and function ofproteins.Genome SegmentProteinLocationProperties & FunctionL1VP1Within the sub-core at theRNA dependent RNA polymerase(3954 bp) (150 kDa)5-fold axisL2VP2Outer capsidOuter capsid, serotype specific(2926 bp) (111 kDa)(trimer)antigen, mammalian cellattachment protein, neutralizingepitopesL3VP3Sub-core capsid layerInnermost protein capsid shell,(2770 bp) (103 kDa)(T = 2 symmetry)sub-core capsid layer, selfassembles, retains icosahedralsymmetry, RNA binding, interactswith internal minor proteinsM4VP4Within the sub-core at theCapping enzyme.(2011 bp) (76 kDa)5-fold axis (dimer)guanylyltransferaseM5VP5Outer capsidInner outer capsid protein, can(1638 bp) (59 kDa)(trimer)affect virus serotype characteristicsM6NS1CytoplasmForms tubules in the cell(1769 bp) (64 kDa)cytoplasmS7VP7Outer coreOuter core surface protein,(1156 bp) (38 kDa)(T = 13 symmetry, trimer)immuno-dominant majorserogroup specific antigen,attachment protein for vectorinsect cells, reacts with ‘coreneutralizing’ antibodiesS8NS2Cytoplasm, viralImportant viral inclusion body(1124 bp) (41 kDa)inclusion bodies (VIB)matrix protein, ssRNA binding,phosphorylated, can be associatedwith outer capsidS9VP6Within the sub-core at thessRNA and dsRNA binding,(1046 bp) (36 kDa)5-fold axishelicase, NTPaseS10NS3,Cell membranesGlycoproteins, membrane proteins,(822 bp) (24 kDa)NS3ainvolved in cell exit
Lobato and Coupar (Lobato, Coupar et al. 1997) developed vaccinia virus-based expression vectors containing various inserts corresponding to nucleotide sequences encoding for structural proteins VP2, VP5 and VP7 of BTV for both in vivo and in vitro studies. These expression vectors were administered to rabbits and sheep to evaluate the immune response with respect to ELISA and neutralizing antibody titer, and the protective efficacy of the VP2 and VP5 constructs was tested in sheep. Vaccinia virus-expressed VP2, VP5 and VP2+VP5 were protective, with the most reproducible protection occurring in animals immunized with both VP2 and VP5 however protection even with this construct was variable.
It would be advantageous to provide improved immunogenic and vaccine compositions against BTV, and methods for making and using such compositions, including such compositions that provide for differential diagnostic methods, assays and kits.
Citation or identification of any document in this application is not admission that such document is available as prior art to the present invention.